[phenixbb] Low resolution refinement

Nathaniel Echols nechols at lbl.gov
Sat Apr 26 07:59:51 PDT 2014

On Fri, Apr 25, 2014 at 3:15 PM, Appu kumar <appu.kumar9 at gmail.com> wrote:

>                  I want to refine the low resolution structure (6.5A). It
> is a membrane protein and we are able to phase it. It will be great help if
> any one can guide me how to proceed further with the refinement. People
> uses Phenix to refine low resolution structure, so phenix is really a good
> program to proceed for refinement against low resolution.

How to proceed depends on what exactly you want the refinement to do.  If
the structure is already approximately correct with no major conformational
changes, here are some basic guidelines:

1) Use tight restraints.  The weight optimization doesn't work very well in
this resolution range (at least in my hands), so setting very low values
for wxc_scale and wxu_scale (which control the weighting of the X-ray term
relative to the geometry and B-factor restraints, respectively) is a good
place to start.  I would try values of wxc_scale=0.025 and wxu_scale=0.05,
but you may need to experiment with this.

2) Use NCS restraints if you have multiple copies in the ASU.  (The torsion
restraints are usually sufficient - and much easier to use - but it might
be worth trying cartesian restraints too.)

3) Use a reference model if you have a good high-resolution structure
available (this can be just part of the structure).

4) Real-space refinement is probably not going to work.

5) Try using a very conservative refinement strategy at first - for
instance, rigid-body and group B-factor refinement.  The latter should work
better now (in the latest nightly builds, and imminent 1.9 release) since
Pavel added restraints on neighboring groups.  You can also try refining
TLS for domains.  My own experience was that individual coordinate and
B-factor refinement worked best - the latter is somewhat controversial but
with tight restraints it ends up not being very different from refining an
overall B-factor.  I also found it helpful to reset all B-factors to 20 at
the start of refinement to remove bias from the starting model.

6) Be very paranoid about the 2mFo-DFc map - at this resolution it will
look almost exactly like your model.  Make lots of omit maps, and if you
have any heavy scatterers (metals, etc.) those will be very helpful in
confirming that you have the correct structure.

If you actually have to do a lot of rebuilding, it is considerably more
difficult; DEN or Rosetta refinement may be helpful.

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